Volume 13 Issue 2 - March 19, 2010 PDF
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Galectin-1, a novel ligand of neuropilin-1, activates VEGFR-2 signaling and modulates the migration of vascular endothelial cells
Institute of Oral Medicine, College of Medicine, National Cheng Kung University
 
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Angiogenesis is a fundamental process required for a number of physiological and pathological events and is considered a key step in tumor growth, invasion, and metastasis. Vascular endothelial growth factor (VEGF), one of the most potent angiogenic cytokines, regulates both vascular proliferation and permeability, and acts as an anti-apoptotic factor for newly formed blood vessels. The biological effects of VEGF are mediated through two major VEGF receptors, VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1). Neuropilin-1 (NRP1) is a transmembrane glycoprotein, interacts with both class 3 semaphorins and VEGF, and is involved in the regulation of many physiological pathways, including angiogenesis. NRP1 also interacts directly with VEGFR-1 and VEGFR-2 and mediates their signal transduction.

Galectin-1 (Gal-1) is a homodimeric animal lectin with a conserved carbohydrate-recognition domain and binding affinity for β-galactosides. Because Gal-1 interacts with specific glycoconjugates, it has been implicated in several biological processes, including cell proliferation, cell adhesion, apoptosis, metastasis, and immunoregulation. Immunohistochemical staining of Gal-1 in clinical cancer samples revealed that Gal-1 expression was positively associated with the malignant progression of many tumor types such as a malignant gliom, prostate cancer, and oral squamous cell carcinoma. Gal-1 was recently identified as being overexpressed in tumor associated capillary endothelial cells, and its expression levels were significantly correlated with tumor aggressiveness. The role of Gal-1 in endothelial cellular functions and the mechanism of action of Gal-1 remain unknown. In this article we first found that Gal-1 was overexpressed in the tumor associated endothelial cells of oral squamous cell carcinomas (Fig. 1). Gal-1 immunostaining in cancer-associated endothelial cells (ECs) was significantly more pronounced than in matched adjacent non-tumor mucosa vascular endothelial cells.
Fig. 1 The expression of galectin-1 (Gal-1) is upregulated in tumor-associated vascular endothelial cells and the surrounding extracellular matrix. Representative immunohistochemical analysis of Gal-1 expression in oral squamous cell carcinoma (OSCC) tissue (b and d), and paired adjacent healthy oral mucosa tissue (a and c). Serial 5 mm histological sections were immunohistochemically stained using polyclonal anti-human Gal-1 antibody. Black arrows and white arrows indicate vascular endothelial cells in normal tissues and OSCC tissues, respectively. Scale bar: 25 μm.

We further investigated the extracellular functions of Gal-1, and explored the molecular pathway by which Gal-1 affects ECs. Gal-1 increased the proliferation and adhesion of endothelial cells, and enhanced cell migration in combination with VEGF165 (Fig. 2a). To better understand the function of Gal-1 and the pathways in which it is involved, we sought to isolate proteins from the cell surface of human umbilical vein endothelial cells (HUVECs) that might interact with Gal-1. Surprisingly, Gal-1 selectively bound NRP1 via the carbohydrate-recognition domain, but did not bind VEGFR-1, VEGFR-2 or VEGFR-3 (Fig. 2b). The Gal-1–NRP1 interaction mediated the migration and adhesion of endothelial cells (Fig 2c, Left). The binding of Gal-1 to NRP1 enhanced VEGFR-2 phosphorylation and stimulated the activation of the mitogen activated protein (MAP) kinases SAPK1/JNK (stress activated protein kinase-1/c-Jun NH2-terminal kinase) (Fig.2c, Right).
Fig. 2 (a) Gal-1 induced HUVECs proliferation and migration. (b) Surface plasmon resonance (SRP) was used to measure real-time association and dissociation of the binding of Gal-1 to VEGFRs. Left, SRP analysis demonstrated that Gal-1 can bind to only NRP1. Right, Gal-1 could specifically bind to NRP1 with high affinity dissociation constant (KD ~0.5×10-7M). (c) Left, Knockdown of NRP1 completely abolished Gal-1-induced HUVEC migration. Right, the activation of the NRP1/VEGFR-2 signaling pathway is required for Gal-1-induced ECs migration.

Taken together, our results indicate that extracellular Gal-1 promotes the proliferation, migration, and adhesion of HUVECs, suggesting an important role for Gal-1 in tumor angiogenesis. Gal-1 functions as an endothelial cell migration enhancer via NRP1 binding, VEGFR-2 activation (Fig 3). The coexpression of Gal-1 and NRP1 may create a novel interacting signal that modulates NRP1-mediated neuron regeneration, angiogenesis, and tumor metastasis. The findings presented here open the way for further investigation of such possibilities, and offer new insights into the role of Gal-1 in NRP1-associated biological processes.
Fig. 3 Summary diagram shows that Gal-1 is a novel ligand of NRP1. Gal-1 binds NRP1 and triggers the NRP1/VEGFR2 signaling pathways, increasing endothelial cell migration and tumor angiogenesis.
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